Adherent cancer cell line expressing a hematological tumor antigen

ABSTRACT

The present invention relates to a transduced cancer cell line stably expressing a leukemia tumor antigen, wherein the cancer cell line is cervical cancer cells, breast cancer cells, ovarian cancer cells, pancreatic cancer cells, lung cancer cells, or glioblastoma cells. The transduced adherent cell line of the present invention is useful for many pre-clinical applications such as real time cytotoxicity assay or to test the effects of CAR-T cells that target the tumor antigen. The present invention is exemplified by Hela cell line stably expressing CD19.

This application is a continuation of U.S. application Ser. No.15/610,419, filed May 31, 2017, which claims the priority of U.S.Provisional Application No. 62/343,976, dated Jun. 1, 2016. The aboveapplications are incorporated herein by reference in their entireties.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing is concurrently submitted herewith with thespecification as an ASCII formatted text file via EFS-Web with a filename of Sequence Listing.txt with a creation date of May 15, 2017, and asize of 13.0 kilobytes. The Sequence Listing filed via EFS-Web is partof the specification and is hereby incorporated in its entirety byreference herein.

FIELD OF THE INVENTION

The present invention relates to Hela-CD19 cell line that stably expressCD19 (cluster of differentiation 19), which is a marker of hematopoieticcancers.

BACKGROUND OF THE INVENTION

Hela cell line was derived from cervical cancer cells taken on Feb. 8,1951 from Henrietta Lacks, who died of cancer on Oct. 4, 1951[Ghorashian et al. Br J Haematol 2015, 169, 463-478.] The cell line wasfound to be remarkably durable and extensively used in scientificresearch.

CD19 is a marker of hematopoietic cancers. B-lymphocyte antigen CD19,also known as CD19 (cluster of differentiation 19), is a protein that inhumans is encoded by the CD19 gene. It is found on the surface ofB-cells and overexpressed in leukemia and lymphoma and effectivelytargeted with CD19-CAR-T cells (Kochenderfer et al., Blood 2013, 122,4129-4139, Scherer et al., J Exp Med 1953, 97, 695-710). There are manyother hematological cancer tumor antigens such as CD4, CD5, CD7, CD8,CD10, CD20, CD22, CD23, CD24, CD33, CD38, CD47, CD56, CD57, CD123,CD138, BCMA and other which can be overexpressed on cancer cell surfacesand can be targeted by target-specific-CAR-T cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the flow cytometry results of the expression of CD19 byHela cells and Hela-CD19 cells.

FIG. 2A shows CD19-CAR-T cells and CD19-FLAG-CAR-T cells effectivelytarget Hela-CD19 line. Effector to target cells ratio is 5:1 in RTCAassay. Hela-CD19 cells are killed by CD19-CAR-T cells andCD19-FLAG-CAR-T cells.

FIG. 2B shows CD19-CAR-NK cells effectively target Hela-CD19 line. NK,Natural Killer cells.

FIG. 3 shows that Hela-CD19 cervical cancer xenograft tumor growth wasblocked by intratumoral injection of CD19-CAR-T cells. Intra-tumoralinjections of CD19-CAR T cells and CD19-FLAG CAR-T cells significantlyinhibited Hela-CD19 tumor growth. The growth curves are shown for thetumors treated with non-transduced T cells and the tumors treated withCD19 (day 19) and CD19-FLAG (day 33) CAR-T cells. *:p<0.0.5 for CAR-Tcells compared to non-transduced T cells, determined by Student's t testassuming unequal variances.

FIG. 4 shows that Hela-CD19 cervical cancer xenograft tumor growth wasblocked by intravenous injection of CD19-FLAG CAR-T cells. Hela-CD19xenograft tumor growth curves, averaged per group, *:p<0.0.05 forCD19-FLAG CAR-T cells compared to non-transduced T cells.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, a “chimeric antigen receptor (CAR)” means a fusedprotein comprising an extracellular domain capable of binding to anantigen, a transmembrane domain derived from a polypeptide differentfrom a polypeptide from which the extracellular domain is derived, andat least one intracellular domain. The “chimeric antigen receptor (CAR)”is sometimes called a “chimeric receptor”, a “T-body”, or a “chimericimmune receptor (CIR).” The “extracellular domain capable of binding toan antigen” means any oligopeptide or polypeptide that can bind to acertain antigen. The “intracellular domain” means any oligopeptide orpolypeptide known to function as a domain that transmits a signal tocause activation or inhibition of a biological process in a cell.

As used herein, a FLAG-tag, or FLAG octapeptide, or FLAG epitope, is apolypeptide protein tag that can be added to a protein using recombinantDNA technology, having the sequence motif DYKDDDDK (SEQ ID NO: 1). Itcan be fused to the C-terminus or the N-terminus of a protein, orinserted within a protein.

As used herein, a “domain” means one region in a polypeptide which isfolded into a particular structure independently of other regions.

As used herein, a “single chain variable fragment (scFv)” means a singlechain polypeptide derived from an antibody which retains the ability tobind to an antigen. An example of the scFv includes an antibodypolypeptide which is formed by a recombinant DNA technique and in whichFv regions of immunoglobulin heavy chain (H chain) and light chain (Lchain) fragments are linked via a spacer sequence. Various methods forpreparing an scFv are known to a person skilled in the art.

As used herein, a “tumor antigen” means a biological molecule havingantigenicity, expression of which causes cancer.

The present invention is directed to a transduced adherent cell linethat stably expresses a leukemia tumor antigen; it is a solid tumor cellline expressing a hematopoietic cancer marker. The adherent cellssuitable in the present invention include those that are negative forhematologic markers and are durable; for example, cervical cancer cellssuch as Hela, breast cancer cells such as MCF-7, ovarian cancer cellssuch as A1747, pancreatic cancer cells such as PANC-1, lung cancer cellssuch as A549, and glioblastoma cells such as U87 or other type of solidcancer (prostate, colon, lung). A preferred cell line is Hela.

The hematological tumor antigen that can be expressed in the transducedadherent cell line of the present invention include CD (Cluster ofDifferentiation)19, CD4, CD5, CD7, CD8, CD10, CD20, CD22, CD23, CD24,CD33, CD38, CD47, CD56, CD57, CD123, CD138, BCMA and other

In one embodiment, the present invention is directed to a transducedHela cell line that stably expresses CD19 (Hela-CD19). The Hela-CD19cell line is greater than 90% positive, preferrably greater than 95%,greater than 98%, or greater than 99%, for expressing CD19.

The transduced adherent cell line of the present invention is useful formany pre-clinical applications. In one embodiment, the transducedadherent cell line is useful for real time cytotoxicity assay withCD19-CAR-T cells, CD2O-CAR-T cells, CD22-CAR-T cells, CD23-CAR-T cells,or CD24-CAR-T cells, or CD4, CD5, CD7, CD8, CD10, CD33, CD38, CD47,CD56, CD57, CD123, CD138, BCMA and other CAR-T cells targeting eachspecific tumor antigen.

The Hela-CD19 cell line can be used in xenograft mice studies to testthe effect of CD19-CAR-T cells.

The transduced cell line of the present invention is a solid cancer cellline that allows to study tumor microenvironment and signaling of solidtumors in vivo. Because it is an adherent cell line, it allows toperform different assays such as RTCA (real time cytotoxicity assay)where adherent properties of cells are used for quantification. Thetransduced cell line expresses tumor markers of leukemia, which can beused to study targeting of leukemia markers and tumor antigens.

The following examples further illustrate the present invention. Theseexamples are intended merely to be illustrative of the present inventionand are not to be construed as being limiting.

EXAMPLES Example 1 Lentiviral Vector Containing Human CD19

The nucleic acid sequence of human CD19 is shown in SEQ ID NO: 2, whichstarts with start codon, ATG and finishes with stop codon TGA. The NCIAccession number of human CD19 is NM_001770. This human CD19 wasinserted into Xba I and EcoR I sites of lentiviral vector (LentiCMV-MCS-EF1a-puro, obtained from Systems Bioscience).

Example 2 Generation of CD19-Encoding Lentivirus and lentivirusProduction

DNAs encoding CD19 cDNA were synthesized and subcloned into athird-generation lentiviral vector, Lenti CMV-MCS-EF1a-puro by SynoBiological (Beijing, China). All lentiviral constructs were sequenced inboth directions to confirm the construct sequence and used forlentivirus production. Ten million growth-arrested HEK293FT cells(Thermo Fisher) were seeded into T75 flasks and cultured overnight, thentransfected with the pPACKH1 Lentivector Packaging mix (SystemBiosciences, Palo Alto, Calif.) and 10 μg of each lentiviral vectorusing the CalPhos Transfection Kit (Takara, Mountain View, Calif.). Thenext day the medium was replaced with fresh medium, and 48 h later thelentivirus-containing medium was collected. The medium was cleared ofcell debris by centrifugation at 2100 g for 30 min. The virus particleswere collected by centrifugation at 112,000 g for 100 min, suspended inDMEM or AIM V medium, aliquoted and frozen at −80° C. The titers of thevirus preparations were determined by quantitative RT-PCR using theLenti-X qRT-PCR kit (Takara) according to the manufacturer's protocoland the 7900HT thermal cycler (Thermo Fisher). The lentiviral titerswere >1×10⁸ pfu/ml.

Example 3 Transduction of Hela Cells with Lentivirus and Generation ofHela-CD19 Cells

CD19-lentivirus of Example 2 were thawed on ice. To each well of 1×10⁶Hela cells, 5×10⁶ CD19-lentivirus, and 2 μL/mL of media of Transplus(Alstem, Richmond, Calif.) (a final dilution of 1:500) were added. Cellswere incubated for an additional 24 hr before repeating addition oflentivirus.

The Hela cells were transduced with lentiviral construct containing CD19DNA, and puromycin was added to the medium to select stable Hela-CD19cells. Puromycin was added 1 μg/ml to DMEM with 10% FBS medium, andfresh medium was changed with puromycin 1 μg/ml every 3 days.

Hela cells were then grown in the continued presence of fresh mediumwith puromycin for a period of 12-14 days. The stable clone of Hela-CD19cells were isolated and grown until confluence. The expression of CD19was confirmed by FACS analysis with CD19 antibody.

The Hela-CD19 stable cell line was received on May 31, 2017, anddeposited on Jun. 1, 2017 with American Type Culture Collection (ATCC)under ATCC deposit number HeLa-CD19, under the Budapest Treatyprovisions.

Example 4 Preparing CAR-T cells (CD19-ScFv-CD28-CD3zeta)

Sequence of CD19-CD28-CD3 zeta was described in [Kochenderfer et al. JImmunother 2009, 32, 689-702]. CD19 scFv and CD28-transmembrane andactivation domains and CD3 zeta were subcloned into a third-generationlentiviral vector, Lenti CMV-MCS-EF1a-puro by Syno Biological (Beijing,China). All CAR lentiviral constructs were sequenced in both directionsto confirm CAR sequence and used for lentivirus production.

The lentiviruses were generated in 293T cells, titer was established byRT-PCR, and was used for transduction of T cells according to protocoldescribed in Example 2 of Berahovich et al Front Biosci (Landmark Ed).22:1644-1654 (2017).

CAR-T cells (CD19ScFv-CD28-CD3zeta) were generated similarly asdescribed for Hela cells in Example 3, but without selection of stablecell clones. No puromycin treatment was performed to preserve highercell number. The CAR-T were used for cytotoxicity assay (Example 5) withtarget Hela-CD19 cells.

Example 5 Cytotoxicity Assay (Real-Time ACEA)

The cytotoxicity was performed using ACEA machine according tomanufacturer's protocol listed below.

Adherent target cells (HeLa or HeLa-CD19) were seeded into 96-wellE-plates (Acea Biosciences, San Diego, Calif.) at 1×10⁴ cells per welland monitored in culture overnight with the impedance-based real-timecell analysis (RTCA) iCELLigence system (Acea Biosciences). The nextday, the medium was removed and replaced with AIM V-AlbuMAX mediumcontaining 10% FBS ±1×10⁵ effector cells (CAR-T cells or non-transducedT cells), in triplicate. The cells in the E-plates were monitored foranother 2-3 days with the RTCA system, and impedance was plotted overtime. Cytolysis was calculated as (impedance of target cells withouteffector cells—impedance of target cells with effectorcells)×100/impedance of target cells without effector cells.

Example 6 Flow Cytometry Results to Detect Expression of CD19

To measure CAR expression, 0.5 million cells were suspended in 100 μl ofbuffer (PBS containing 0.5% BSA) and incubated on ice with 1 μl of humanserum (Jackson Immunoresearch, West Grove, Pa.) for 10 min. Theanti-CD19-PE or its isotype control PE-labelled antibody were added, andthe cells were incubated on ice for 30 min. The cells were rinsed with 3ml of buffer, then suspended in buffer and acquired on a FACSCalibur (BDBiosciences).

Hela cells are negative for CD19. In Example 3, the Hela cells werestably transduced with CD19, and the resulting Hela-CD19 cells expressCD19, as verified by flow cytometry with CD19-PE antibody (FIG. 1). Thecells were almost 99% positive for CD19.

Example 7 Hela-CD19 Cells are Targeted by CD19-CAR-T and CD19/NK CellsIn Vitro

We used Hela-CD19 cells (Example 3), T cells, and CD19-CD28-CD3-zetaCAR-T (CD19scFv CAR-T, Example 4) cells in this Experiment.

FIG. 2A shows real-time cytotoxicity assay with Hela-CD19 target cellsand CD19-CAR-T effector cells. FIG. 2B shows real-time cytotoxicityassay with Hela-CD19 target cells and CD19-CAR effector NK cells.

FIG. 2A demonstrates that CD19-CAR-T cells effectively target Hela-CD19line. The CD19-CAR-T cells were CD19-CD28-CD3 zeta, second generationCAR-T cells. Thus, Hela-CD19 can be used in cytotoxicity assays withCD19-CAR-T cells targeting CD19 antigen.

The same result was obtained with CD19-NK (natural killer) cells (FIG.2B).

Example 8 Hela-CD19 Cells are Targeted by CD19-CAR-T Cells In Vivo

To test that Hela-CD19 cells can be used with hematological cancertargets, we developed a novel xenograft tumor model using the HeLa-CD19cell line. Immunodeficient NSG mice were injected subcutaneously on eachflank with 2×10⁶ HeLa-CD19 cells, and the sizes of the tumors weremonitored for 36 days. The tumors injected intratumorally with CD19 andCD19-FLAG CAR-T cells (average 285 mm³) were significantly smaller thanthe control tumors injected with non-transduced T cells (average 935mm³). FIG. 3 shows the average growth curves for the tumors treated withnon-transduced T cells and the tumors treated with CD19 CAR-T cells onday 19 and CD19-FLAG CAR-T cells on day 33. The results show thatintra-tumoral injections of CD19 and CD19-FLAG CAR-T cells significantlyinhibited HeLa-CD19 tumor growth. *:p<0.05 for CAR-T cells compared tonon-transduced T cells, determined by Student's t test assuming unequalvariances.

To characterize the effect of CD19-FLAG CAR-T cells in the HeLa-CD19solid tumor model, a second study was conducted with earlier,intravenous application of the CD19-FLAG CAR-T cells. In this study, theCD19-FLAG CAR-T cells almost completely blocked tumor growth (see FIG.4)

The results show that solid cancer Hela cell line with stablyoverexpressed leukemia antigen can be used as a model of solid cancer tostudy hematological targets in the solid tumor microenvironment allowingto study solid tumor biology, signaling and effect of hematologicalmarket targeting.

Example 9 CD20 Nucleotide Sequence

Examples 9-12 shows the nucleotide sequences of CD20, CD22, CD23, andCD24. The same approach as described above are used to generate solidtumor cell lines such as stable Hela or other stable cancer cell lines,which stably expressing these leukemia tumor antigens.

CD20 (Membrane-Spanning 4-Domains Subfamily A Member 1), nucleotidesequence is shown as SEQ ID NO: 4 (NCBI Accesion number, NM_021950.3),

Example 10 CD22 Nucleotide Sequence

CD22, nucleotide sequence is shown as SEQ ID NO: 5; Gen Bank AccessionNumber, X52785.1.

Example 11 CD23 Nucleotide Sequence

CD23 (other name: Fc Fragment Of IgE, Low Affinity II, Receptor For(CD23) is shown as SEQ ID NO: 6; Accesion number: AC008763.

Example 12 CD24 Nucleotide Sequence

CD24, (Accession NCBI number: NM_001291737.1

NM_001291738.1, NM_001291739.1, NM_013230.3, SEQ ID NO: 7

Example 13 Other Hematological Cancer Antigens Nucleotide and Amino-AcidSequences

CD4, CD5, CD7, CD8, CD10, CD33, CD38, CD47, CD56, CD57, CD123, CD138,BCMA and other nucleotide sequences are available from NCBI or Uniprotdatabases, respectively (www.uniprot.org).

The same approach as described above are used to generate solid tumorcell lines such as stable Hela or other stable cancer cell lines, whichstably expressing these leukemia tumor antigens.

It is to be understood that the foregoing describes preferredembodiments of the present invention and that modifications may be madetherein without departing from the scope of the present invention as setforth in the claims.

What is claimed is:
 1. A method for measuring cytotoxicity of CD19-CAR Tcells, comprising: seeding transduced Hela cancer cells that stablyexpress CD19 on a plate, adding CD19-CAR T cells to the plate, andmeasuring real time cytotoxicity of the CD19-CAR T cells.
 2. The methodof claim 1, wherein the CAR comprises CD19 scFv, CD28-transmembranedomain, CD28 activation domain, and CD3 zeta.
 3. The method of claim 1,wherein the CAR further comprises a FLAG tag.